Biosynthetic proteins targeting the SARS-CoV-2 spike as anti-virals.

The binding of the SARS-CoV-2 spike to angiotensin-converting enzyme 2 (ACE2) promotes virus entry into the cell. Targeting this interaction represents a promising strategy to generate antivirals. By screening a phage-display library of biosynthetic protein sequences build on a rigid alpha-helicoidal HEAT-like scaffold (named αReps), we selected candidates recognizing the spike receptor binding domain (RBD). Two of them (F9 and C2) bind the RBD with affinities in the nM range, displaying neutralisation activity in vitro and recognizing distinct sites, F9 overlapping the ACE2 binding motif. The F9-C2 fusion protein and a trivalent αRep form (C2-foldon) display 0.1 nM affinities and EC50 of 8-18 nM for neutralization of SARS-CoV-2. In hamsters, F9-C2 instillation in the nasal cavity before or during infections effectively reduced the replication of a SARS-CoV-2 strain harbouring the D614G mutation in the nasal epithelium. Furthermore, F9-C2 and/or C2-foldon effectively neutralized SARS-CoV-2 variants (including delta and omicron variants) with EC50 values ranging from 13 to 32 nM. With their high stability and their high potency against SARS-CoV-2 variants, αReps provide a promising tool for SARS-CoV-2 therapeutics to target the nasal cavity and mitigate virus dissemination in the proximal environment.

SARS-CoV-2 Spike Furin Cleavage Site and S2' Basic Residues Modulate the Entry Process in a Host Cell-Dependent Manner.

SARS-CoV-2 spike (S) envelope glycoprotein constitutes the main determinant of virus entry and the target of host immune response, thus being of great interest for antiviral research. It is constituted of S1 and S2 subunits, which are involved in ACE2 receptor binding and fusion between the viral envelope and host cell membrane, respectively. Induction of the fusion process requires S cleavage at the S1-S2 junction and the S2' site located upstream of the fusion peptide. Interestingly, the SARS-CoV-2 spike harbors a 4-residue insertion at the S1-S2 junction that is absent in its closest relatives and constitutes a polybasic motif recognized by furin-like proteases. In addition, the S2' site is characterized by the presence of conserved basic residues. Here, we sought to determine the importance of the furin cleavage site (FCS) and the S2' basic residues for S-mediated entry functions. We determined the impact of mutations introduced at these sites on S processing, fusogenic activity, and its ability to mediate entry in different cellular backgrounds. Strikingly, mutation phenotypes were highly dependent on the host cell background. We confirmed that although the FCS was not absolutely required for virus entry, it contributed to extending the fusogenic potential of S. Cleavage site mutations, as well as inhibition of furin protease activity, affected the cell surface expression of S in a host cell-dependent manner. Finally, inhibition of furin activity differentially affected SARS-CoV-2 virus infectivity in the tested host cells, thereby confirming the host cell-dependent effect of spike processing for the viral life cycle. IMPORTANCE SARS-CoV-2 is responsible for the current global pandemic that has resulted in several million deaths. As the key determinant of virus entry into host cells and the main target of host immune response, the spike glycoprotein constitutes an attractive target for therapeutics development. Entry functions of spike rely on its processing at two sites by host cell proteases. While SARS-CoV-2 spike differs from its closest relatives by the insertion of a basic furin cleavage motif at the first site, it harbors conserved basic residues at the second cleavage site. Characterization of the importance of the basic sequences present at the two cleavage sites revealed that they were influencing spike processing, intracellular localization, induction of fusion, and entry in a host cell-dependent manner. Thus, our results revealed a high heterogeneity in spike sequence requirement for entry functions in the different host cells, in agreement with the high adaptability of the SARS-CoV-2 virus.

Role of the cytosolic domain of occludin in trafficking and hepatitis C virus infection.

The role of the tight-junction (TJ) protein occludin (OCLN) in hepatitis C virus (HCV) entry remains elusive. Here, we investigated the OCLN C-terminal cytosolic domain in HCV infection. We expressed a series of C-terminal deletion mutants in Huh-7 cells KO for OCLN and characterized their functionality in HCV infection and trafficking. Deleting the OCLN cytosolic domain led to protein instability and intracellular retention. The first 15 residues (OCLN-C15 mutant) of the cytosolic domain were sufficient for OCLN stability, but led to its accumulation in the trans-Golgi network (TGN) due to a deficient cell surface export after synthesis. In contrast, the OCLN-C18 mutant, containing the first 18 residues of the cytosolic domain, was expressed at the cell surface and could mediate HCV infection. Point mutations in the context of C18 showed that I279 and W281 are crucial residues for cell surface expression of OCLN-C18. However, in the context of full-length OCLN, mutation of these residues only partially affected infection and cell surface localization. Importantly, the characterization of OCLN-C18 in human-polarized hepatocytes revealed a defect in its TJ localization without affecting HCV infection. These data suggest that TJ localization of OCLN is not a prerequisite for HCV infection in polarized hepatocytes.

Dehydrojuncusol, a Natural Phenanthrene Compound Extracted from Juncus maritimus, Is a New Inhibitor of Hepatitis C Virus RNA Replication.

Recent emergence of direct-acting antivirals (DAAs) targeting hepatitis C virus (HCV) proteins has considerably enhanced the success of antiviral therapy. However, the appearance of DAA-resistant-associated variants is a cause of treatment failure, and the high cost of DAAs renders the therapy not accessible in countries with inadequate medical infrastructures. Therefore, the search for new inhibitors with a lower cost of production should be pursued. In this context, the crude extract of Juncus maritimus Lam. was shown to exhibit high antiviral activity against HCV in cell culture. Bio-guided fractionation allowed the isolation and identification of the active compound, dehydrojuncusol. A time-of-addition assay showed that dehydrojuncusol significantly inhibited HCV infection when added after virus inoculation of HCV genotype 2a (50% effective concentration [EC50] = 1.35 µM). This antiviral activity was confirmed with an HCV subgenomic replicon, and no effect on HCV pseudoparticle entry was observed. Antiviral activity of dehydrojuncusol was also demonstrated in primary human hepatocytes. No in vitro toxicity was observed at active concentrations. Dehydrojuncusol is also efficient on HCV genotype 3a and can be used in combination with sofosbuvir. Interestingly, dehydrojuncusol was able to inhibit RNA replication of two frequent daclatasvir-resistant mutants (L31M or Y93H in NS5A). Finally, mutants resistant to dehydrojuncusol were obtained and showed that the HCV NS5A protein is the target of the molecule. In conclusion, dehydrojuncusol, a natural compound extracted from J. maritimus, inhibits infection of different HCV genotypes by targeting the NS5A protein and is active against resistant HCV variants frequently found in patients with treatment failure.IMPORTANCE Tens of millions of people are infected with hepatitis C virus (HCV) worldwide. Recently marketed direct-acting antivirals (DAAs) targeting HCV proteins have enhanced the efficacy of treatment. However, due to its high cost, this new therapy is not accessible to the vast majority of infected patients. Furthermore, treatment failures have also been reported due to the appearance of viral resistance. Here, we report on the identification of a new HCV inhibitor, dehydrojuncusol, that targets HCV NS5A and is able to inhibit RNA replication of replicons harboring resistance mutations to anti-NS5A DAAs used in current therapy. Dehydrojuncusol is a natural compound isolated from Juncus maritimus, a halophilic plant species that is very common in coastlines worldwide. This molecule might serve as a lead for the development of a new therapy that is more accessible to hepatitis C patients in the future.

Functional Study of the C-Terminal Part of the Hepatitis C Virus E1 Ectodomain.

In the hepatitis C virus (HCV) envelope glycoproteins E1 and E2, which form a heterodimer, E2 is the receptor binding protein and the major target of neutralizing antibodies, whereas the function of E1 remains less characterized. To investigate E1 functions, we generated a series of mutants in the conserved residues of the C-terminal region of the E1 ectodomain in the context of an infectious clone. We focused our analyses on two regions of interest. The first region is located in the middle of the E1 glycoprotein (between amino acid [aa] 270 and aa 291), which contains a conserved hydrophobic sequence and was proposed to constitute a putative fusion peptide. The second series of mutants was generated in the region from aa 314 to aa 342 (the aa314-342 region), which has been shown to contain two α helices (α2 and α3) by nuclear magnetic resonance studies. Of the 22 generated mutants, 20 were either attenuated or noninfectious. Several mutations modulated the virus's dependence on claudin-1 and the scavenger receptor BI coreceptors for entry. Most of the mutations in the putative fusion peptide region affected virus assembly. Conversely, mutations in the α-helix aa 315 to 324 (315-324) residues M318, W320, D321, and M322 resulted in a complete loss of infectivity without any impact on E1E2 folding and on viral assembly. Further characterization of the W320A mutant in the HCVpp model indicated that the loss of infectivity was due to a defect in viral entry. Together, these results support a role for E1 in modulating HCV interaction with its coreceptors and in HCV assembly. They also highlight the involvement of α-helix 315-324 in a late step of HCV entry.IMPORTANCE HCV is a major public health problem worldwide. The virion harbors two envelope proteins, E1 and E2, which are involved at different steps of the viral life cycle. Whereas E2 has been extensively characterized, the function of E1 remains poorly defined. We characterized here the function of the putative fusion peptide and the region containing α helices of the E1 ectodomain, which had been previously suggested to be important for virus entry. We could confirm the importance of these regions for the virus infectivity. Interestingly, we found several residues modulating the virus's dependence on several HCV receptors, thus highlighting the role of E1 in the interaction of the virus with cellular receptors. Whereas mutations in the putative fusion peptide affected HCV infectivity and morphogenesis, several mutations in the α2-helix region led to a loss of infectivity with no effect on assembly, indicating a role of this region in virus entry.

Identification of Novel Functions for Hepatitis C Virus Envelope Glycoprotein E1 in Virus Entry and Assembly.

Hepatitis C virus (HCV) envelope glycoprotein complex is composed of E1 and E2 subunits. E2 is the receptor-binding protein as well as the major target of neutralizing antibodies, whereas the functions of E1 remain poorly defined. Here, we took advantage of the recently published structure of the N-terminal region of the E1 ectodomain to interrogate the functions of this glycoprotein by mutating residues within this 79-amino-acid region in the context of an infectious clone. The phenotypes of the mutants were characterized to determine the effects of the mutations on virus entry, replication, and assembly. Furthermore, biochemical approaches were also used to characterize the folding and assembly of E1E2 heterodimers. Thirteen out of 19 mutations led to viral attenuation or inactivation. Interestingly, two attenuated mutants, T213A and I262A, were less dependent on claudin-1 for cellular entry in Huh-7 cells. Instead, these viruses relied on claudin-6, indicating a shift in receptor dependence for these two mutants in the target cell line. An unexpected phenotype was also observed for mutant D263A which was no longer infectious but still showed a good level of core protein secretion. Furthermore, genomic RNA was absent from these noninfectious viral particles, indicating that the D263A mutation leads to the assembly and release of viral particles devoid of genomic RNA. Finally, a change in subcellular colocalization between HCV RNA and E1 was observed for the D263A mutant. This unique observation highlights for the first time cross talk between HCV glycoprotein E1 and the genomic RNA during HCV morphogenesis.IMPORTANCE Hepatitis C virus (HCV) infection is a major public health problem worldwide. It encodes two envelope proteins, E1 and E2, which play a major role in the life cycle of this virus. E2 has been extensively characterized, whereas E1 remains poorly understood. Here, we investigated E1 functions by using site-directed mutagenesis in the context of the viral life cycle. Our results identify unique phenotypes. Unexpectedly, two mutants clearly showed a shift in receptor dependence for cell entry, highlighting a role for E1 in modulating HCV particle interaction with a cellular receptor(s). More importantly, another mutant led to the assembly and release of viral particles devoid of genomic RNA. This unique phenotype was further characterized, and we observed a change in subcellular colocalization between HCV RNA and E1. This unique observation highlights for the first time cross talk between a viral envelope protein and genomic RNA during morphogenesis.

Identification of a New Benzimidazole Derivative as an Antiviral against Hepatitis C Virus.

UNLABELLED: Aminoquinolines and piperazines, linked or not, have been used successfully to treat malaria, and some molecules of this family also exhibit antiviral properties. Here we tested several derivatives of 4-aminoquinolines and piperazines for their activity against hepatitis C virus (HCV). We screened 11 molecules from three different families of compounds, and we identified anti-HCV activity in cell culture for six of them. Of these, we selected a compound (B5) that is currently ending clinical phase I evaluation for neurodegenerative diseases. In hepatoma cells, B5 inhibited HCV infection in a pangenotypic and dose-dependent manner, and its antiviral activity was confirmed in primary hepatocytes. B5 also inhibited infection by pseudoparticles expressing HCV envelope glycoproteins E1 and E2, and we demonstrated that it affects a postattachment stage of the entry step. Virus with resistance to B5 was selected by sequential passage in the presence of the drug, and reverse genetics experiments indicated that resistance was conferred mainly by a single mutation in the putative fusion peptide of E1 envelope glycoprotein (F291I). Furthermore, analyses of the effects of other closely related compounds on the B5-resistant mutant suggest that B5 shares a mode of action with other 4-aminoquinoline-based molecules. Finally, mice with humanized liver that were treated with B5 showed a delay in the kinetics of the viral infection. In conclusion, B5 is a novel interesting anti-HCV molecule that could be used to decipher the early steps of the HCV life cycle. IMPORTANCE: In the last 4 years, HCV therapy has been profoundly improved with the approval of direct-acting antivirals in clinical practice. Nevertheless, the high costs of these drugs limit access to therapy in most countries. The present study reports the identification and characterization of a compound (B5) that inhibits HCV propagation in cell culture and is currently ending clinical phase I evaluation for neurodegenerative diseases. This molecule inhibits the HCV life cycle by blocking virus entry. Interestingly, after selection of drug-resistant virus, a resistance mutation in the putative fusion peptide of E1 envelope glycoprotein was identified, indicating that B5 could be used to further investigate the fusion mechanism. Furthermore, mice with humanized liver treated with B5 showed a delay in the kinetics of the viral infection. In conclusion, B5 is a novel interesting anti-HCV molecule that could be used to decipher the early steps of the HCV life cycle.

Identification of conserved residues in hepatitis C virus envelope glycoprotein E2 that modulate virus dependence on CD81 and SRB1 entry factors.

UNLABELLED: In spite of the high variability of its sequence, hepatitis C virus (HCV) envelope glycoprotein E2 contains several conserved regions. In this study, we explored the structural and functional features of the highly conserved E2 segment from amino acid (aa) 502 to 520, which had been proposed as a fusion peptide and shown to strongly overlap a potential conserved neutralizing epitope. For this purpose, we used reverse genetics to introduce point mutations within this region, and we characterized the phenotypes of these mutants in the light of the recently published structure of E2. The functional analyses showed that their phenotypes are in agreement with the positions of the corresponding residues in the E2 crystal structure. In contrast, our data ruled out the involvement of this region in membrane fusion, and they indicate that alternative conformations would be necessary to expose the potential neutralizing epitope present in this segment. Of particular interest, we identified three specific mutations (Y507L, V514A, and V515A) located within this neutralizing epitope which only mildly reduced infectivity and showed no assembly defect. These mutations modulated HCV dependence on the viral receptor SRB1, and/or they also modulated virion sensitivity to neutralizing antibodies. Importantly, their characterization also showed that amino acids Y507, V514, and V515 contribute to E2 interaction with HCV receptor CD81. In conclusion, our data show that the highly conserved E2 segment from aa 502 to 520 plays a key role in cell entry by influencing the association of the viral particle with coreceptors and neutralizing antibodies. IMPORTANCE: Hepatitis C virus (HCV) envelope proteins E1 and E2 exhibit sequence variability. However, some segments of the envelope proteins are highly conserved, suggesting that these sequences play a key role at some steps of the HCV life cycle. In this work, we characterized the function and structure of a highly conserved E2 region that is targeted by neutralizing antibodies and had been proposed as a fusion peptide. Our data ruled out the involvement of this region in membrane fusion but allowed for the identification of new residues modulating the interaction of the virus with entry factors and its sensitivity to neutralizing antibodies. Moreover, structural data suggest that alternative conformations could exist for E2, which would explain the presence of a partially masked neutralizing epitope in this segment in the currently available E2 structure. Overall, our findings highlight the importance of conserved regions in the sequences of HCV envelope proteins.

Antitumoral activity of parvovirus-mediated IL-2 and MCP-3/CCL7 delivery into human pancreatic cancer: implication of leucocyte recruitment.

Pancreatic ductal adenocarcinoma (PDAC) represents the fourth leading cause of cancer-related death in western countries. The patients are often diagnosed in advanced metastatic stages, and the prognosis remains extremely poor with an overall 5-year survival rate less than 5 %. Currently, novel therapeutic strategies are being pursued to combat PDAC, including oncolytic viruses, either in their natural forms or armed with immunostimulatory molecules. Natural killer cells are critical players against tumours and infected cells. Recently, we showed that IL-2-activated human NK cells displayed killing activity against PDAC cells, which could further be enhanced through the infection of PDAC cells with the rodent parvovirus H-1PV. In this study, the therapeutic efficacy of parvovirus-mediated delivery of three distinct cyto/chemokines (Il-2, MCP-3/CCL7 and IP-10/CXCL10) was evaluated in xenograft models of human PDAC. We show here that activated NK and monocytic cells were found to be recruited by PDAC tumours upon infection with parvoviruses armed with IL-2 or the chemokine MCP-3/CCL7, resulting in a strong anti-tumour response.

A reporter cell line for the automated quantification of SARS-CoV-2 infection in living cells.

The SARS-CoV-2 pandemic and the urgent need for massive antiviral testing highlighted the lack of a good cell-based assay that allowed for a fast, automated screening of antivirals in high-throughput content with minimal handling requirements in a BSL-3 environment. The present paper describes the construction of a green fluorescent substrate that, upon cleavage by the SARS-CoV-2 main protease, re-localizes from the cytoplasm in non-infected cells to the nucleus in infected cells. The construction was stably expressed, together with a red fluorescent nuclear marker, in a highly susceptible clone derived from Vero-81 cells. With this fluorescent reporter cell line, named F1G-red, SARS-CoV-2 infection can be scored automatically in living cells by comparing the patterns of green and red fluorescence signals acquired by automated confocal microscopy in a 384-well plate format. We show the F1G-red system is sensitive to several SARS-CoV-2 variants of concern and that it can be used to assess antiviral activities of compounds in dose-response experiments. This high-throughput system will provide a reliable tool for antiviral screening against SARS-CoV-2.

The association of CD81 with tetraspanin-enriched microdomains is not essential for Hepatitis C virus entry.

BACKGROUND: Three percent of the world's population is chronically infected with hepatitis C virus (HCV) and thus at risk of developing liver cancer. Although precise mechanisms regulating HCV entry into hepatic cells are still unknown, several cell surface proteins have been identified as entry factors for this virus. Among these molecules, the tetraspanin CD81 is essential for HCV entry. Interestingly, CD81 is also required for Plasmodium infection. A major characteristic of tetraspanins is their ability to interact with each other and other transmembrane proteins to build tetraspanin-enriched microdomains (TEM). RESULTS: In our study, we describe a human hepatoma Huh-7 cell clone (Huh-7w7) which has lost CD81 expression and can be infected by HCV when human CD81 (hCD81) or mouse CD81 (mCD81) is ectopically expressed. We took advantage of these permissive cells expressing mCD81 and the previously described MT81/MT81w mAbs to analyze the role of TEM-associated CD81 in HCV infection. Importantly, MT81w antibody, which only recognizes TEM-associated mCD81, did not strongly affect HCV infection. Furthermore, cholesterol depletion, which inhibits HCV infection and reduces total cell surface expression of CD81, did not affect TEM-associated CD81 levels. In addition, sphingomyelinase treatment, which also reduces HCV infection and cell surface expression of total CD81, raised TEM-associated CD81 levels. CONCLUSION: In contrast to Plasmodium infection, our data show that association of CD81 with TEM is not essential for the early steps of HCV life cycle, indicating that these two pathogens, while using the same molecules, invade their host by different mechanisms.

Ceramide enrichment of the plasma membrane induces CD81 internalization and inhibits hepatitis C virus entry.

Virus entry is a major step in which host-cell lipids can play an essential role. In this report, we investigated the importance of sphingolipids in hepatitis C virus (HCV) entry. For this purpose, sphingomyelin present in the plasma membrane of target cells was hydrolysed into ceramide by sphingomyelinase treatment. Interestingly, ceramide enrichment of the plasma membrane strongly inhibited HCV entry. To understand how ceramide affected HCV entry, we analysed the effect of ceramide enrichment of the plasma membrane on three cell-surface molecules identified as entry factors for HCV: CD81 tetraspanin, scavenger receptor BI and Claudin-1. These proteins, which we found to be mainly associated with detergent-soluble membranes in Huh-7 cells, were not relocated in detergent-resistant microdomains after sphingomyelin hydrolysis into ceramide. Importantly, ceramide enrichment of the plasma membrane led to a 50% decrease in cell-surface CD81, which was due to its ATP-independent internalization. Our results strongly suggest that the ceramide-induced internalization of CD81 is responsible for the inhibitory effect of ceramide on HCV entry. Together, these data indicate that some specific lipids of the plasma membrane are essential for HCV entry and highlight plasma membrane lipids as potential targets to block HCV entry.

Cytokines and serum amyloid A in the pathogenesis of hepatitis C virus infection.

Expression of the acute phase protein serum amyloid A (SAA) is dependent on the release of the pro-inflammatory cytokines IL-1, IL-6 and TNF-α during infection and inflammation. Hepatitis C virus (HCV) upregulates SAA-inducing cytokines. In line with this, a segment of chronically infected individuals display increased circulating levels of SAA. SAA has even been proposed to be a potential biomarker to evaluate treatment efficiency and the course of disease. SAA possesses antiviral activity against HCV via direct interaction with the viral particle, but might also divert infectivity through its function as an apolipoprotein. On the other hand, SAA shares inflammatory and angiogenic activity with chemotactic cytokines by activating the G protein-coupled receptor, formyl peptide receptor 2. These latter properties might promote chronic inflammation and hepatic injury. Indeed, up to 80 % of infected individuals develop chronic disease because they cannot completely clear the infection, due to diversion of the immune response. In this review, we summarize the interconnection between SAA and cytokines in the context of HCV infection and highlight the dual role SAA could play in this disease. Nevertheless, more research is needed to establish whether the balance between those opposing activities can be tilted in favor of the host defense.

Glycan Shielding and Modulation of Hepatitis C Virus Neutralizing Antibodies.

Hepatitis C virus (HCV) envelope glycoprotein heterodimer, E1E2, plays an essential role in virus entry and assembly. Furthermore, due to their exposure at the surface of the virion, these proteins are the major targets of anti-HCV neutralizing antibodies. Their ectodomain are heavily glycosylated with up to 5 sites on E1 and up to 11 sites on E2 modified by N-linked glycans. Thus, one-third of the molecular mass of E1E2 heterodimer corresponds to glycans. Despite the high sequence variability of E1 and E2, N-glycosylation sites of these proteins are generally conserved among the seven major HCV genotypes. N-glycans have been shown to be involved in E1E2 folding and modulate different functions of the envelope glycoproteins. Indeed, site-directed mutagenesis studies have shown that specific glycans are needed for virion assembly and infectivity. They can notably affect envelope protein entry functions by modulating their affinity for HCV receptors and their fusion activity. Importantly, glycans have also been shown to play a key role in immune evasion by masking antigenic sites targeted by neutralizing antibodies. It is well known that the high mutational rate of HCV polymerase facilitates the appearance of neutralization resistant mutants, and occurrence of mutations leading to glycan shifting is one of the mechanisms used by this virus to escape host humoral immune response. As a consequence of the importance of the glycan shield for HCV immune evasion, the deletion of N-glycans also leads to an increase in E1E2 immunogenicity and can induce a more potent antibody response against HCV.

Interplay between hepatitis C virus and lipid metabolism during virus entry and assembly.

Hepatitis C virus (HCV) infection is a major public health problem worldwide. In most cases, HCV infection becomes chronic, leading to the development of liver diseases that range from fibrosis to cirrhosis and hepatocellular carcinoma. Due to its medical importance, the HCV life cycle has been deeply characterized, and a unique feature of this virus is its interplay with lipids. Accordingly, all the steps of the virus life cycle are influenced by the host lipid metabolism. Indeed, due to their association with host lipoproteins, HCV particles have a unique lipid composition. Furthermore, the biogenesis pathway of very low density lipoproteins has been shown to be involved in HCV morphogenesis with apolipoprotein E being an essential element for the production of infectious HCV particles. Association of viral components with host cytoplasmic lipid droplets is also central to the HCV morphogenesis process. Finally, due to its close connection with host lipoproteins, HCV particle also uses several lipoprotein receptors to initiate its infectious cycle. In this review, we outline the way host lipoproteins participate to HCV particle composition, entry and assembly.

Recombinant Parvoviruses Armed to Deliver CXCL4L1 and CXCL10 Are Impaired in Their Antiangiogenic and Antitumoral Effects in a Kaposi Sarcoma Tumor Model Due To the Chemokines' Interference with the Virus Cycle.

Application of oncolytic viruses is a valuable option to broaden the armament of anticancer therapies, as these combine specific cytotoxic effects and immune-stimulating properties. The self-replicating H-1 parvovirus (H-1PV) is a prototypical oncolytic virus that, besides targeting tumor cells, also infects endothelial cells, thus combining oncolytic and angiostatic traits. To increase its therapeutic value, H-1PV can be armed with cytokines or chemokines to enhance the immunological response. Some chemokines-more specifically, the CXCR3 ligands CXCL4L1 and CXCL10-combine immune-stimulating properties with angiostatic activity. This study explores the therapeutic value of recombinant parvoviruses carrying CXCL4L1 or CXCL10 transgenes (Chi-H1/CXCL4L1 or Chi-H1/CXCL10, respectively) to inhibit the growth of the human Kaposi sarcoma cell line KS-IMM. KS-IMM cells infected by Chi-H1/CXCL4L1 or Chi-H1/CXCL10 released the corresponding chemokine and showed reduced migratory capacity. Therefore, the antitumoral capacity of Chi-H1/CXCL4L1 or Chi-H1/CXCL10 was tested in mice. Either in vitro infected KS-IMM cells were injected or subcutaneously growing KS-IMM xenografts were treated by peritumoral injections of the different viruses. Surprisingly, the transgenes did not increase the antitumoral effect of natural H-1PV. Further experiments indicated that CXCL4L1 and CXCL10 interfered with the expression of the viral NS1 protein in KS-IMM cells. These results indicate that the outcome of parvovirus-based delivery of CXCR3 ligands might be tumor cell type dependent, and hence its application must be considered carefully.

Distribution and sequence analysis of a family of type ill-dependent effectors correlate with the phylogeny of Ralstonia solanacearum strains.

In Ralstonia solanacearum, we previously have reported on the characterization of popP1 and popP2 genes. These genes encode type III-dependent pathogenicity effectors related to the large family of AvrRxv/YopJ cysteine proteases that are shared among pathogens of plants and animals. In this study, we identify a third gene, named popP3, that is inactivated in the genome sequence of strain GMI1000 by insertion of a copy of the insertion sequence ISRso13. The three popP genes are localized on two large chromosomal pathogenicity islands, with popP1 and popP2 being present on the same island. Phylogenic analysis demonstrated that the PopP2 and PopP3 proteins are clearly distinct from other effectors of this family previously characterized in plant and animal pathogens. Analysis of the distribution and allelic variations of the three genes in 30 strains representative of the biodiversity of R. solanacearum established that popP genes are distributed widely among strains from two of the three phyla previously defined on the basis of the structure of the core genome. Sequencing of the popP genes from the different strains revealed limited allelic variations at the three loci but did not show evidence of recombination between the popP genes. Limited allelic variation together with occurrence of insertion sequences within or in the close vicinity of popP genes and the presence of gene duplications in these pathogenicity islands suggest that genomic rearrangements might be a major evolutionary driving force controlling evolution of the genes encoded in these regions. The implications of these observations in terms of bacterial evolution, gene acquisition, and horizontal gene transfers are discussed.

PopP1, a new member of the YopJ/AvrRxv family of type III effector proteins, acts as a host-specificity factor and modulates aggressiveness of Ralstonia solanacearum.

A functional analysis of an 11-kb-long region of the genome of the plant-pathogenic bacterium Ralstonia solanacearum, previously identified as an alternative codon usage region (ACUR), reveals that it was probably acquired through horizontal gene transfer. This ACUR encodes an insertion sequence and eight potential proteins, one of which is partially homologous with a host-specificity factor from a plant-pathogenic Erwinia sp., and another, PopP1, which is homologous to members of the YopJ/AvrRxv family of type III-secreted bacterial effectors controlling interaction between bacteria and their hosts. The analysis of mutants affecting all except one of the genes identified in the ACUR showed that only the popP1-deficient strain had an altered phenotype in plant infection tests. This mutant strain became pathogenic to a Petunia line that is resistant to the wild-type strain. Therefore, popP1 behaves as a typical avirulence gene. We demonstrate that PopP1 protein is secreted and that secretion of this protein requires a functional type III-secretion pathway. In contrast to the structural genes for other type III-secreted proteins identified in R. solanacearum, transcription of the popP1 gene is not coregulated with transcription of hrp genes but is constitutive.

Capacity of wild-type and chemokine-armed parvovirus H-1PV for inhibiting neo-angiogenesis.

Anti-angiogenic therapy has been recognized as a powerful potential strategy for impeding the growth of various tumors. However no major therapeutic effects have been observed to date, mainly because of the emergence of several resistance mechanisms. Among novel strategies to target tumor vasculature, some oncolytic viruses open up new prospects. In this context, we addressed the question whether the rodent parvovirus H-1PV can target endothelial cells. We show that cultures of human normal (HUVEC) and immortalized (KS-IMM) endothelial cells sustain an abortive viral cycle upon infection with H-1PV and are sensitive to H-1PV cytotoxicity. H-1PV significantly inhibits infected KS-IMM tumor growth. This effect may be traced back by the virus ability to both kill proliferating endothelial cells and inhibit VEGF production Recombinant H-1PV vectors can also transduce tumor cells with chemokines endowed with anti-angiogenesis properties, and warrant further validation for the treatment of highly vascularized tumors.

Assembly of a functional HCV glycoprotein heterodimer.

The two HCV envelope glycoproteins E1 and E2 are released from HCV polyprotein by signal peptidase cleavages. These glycoproteins are type I transmembrane proteins with a highly glycosylated N-terminal ectodomain and a C-terminal hydrophobic anchor. After their synthesis, HCV glycoproteins E1 and E2 associate as a noncovalent heterodimer. The transmembrane domains of HCV envelope glycoproteins play a major role in E1E2 heterodimer assembly and subcellular localization. The envelope glycoprotein complex E1E2 has been proposed to be essential for HCV entry. However, for a long time, HCV entry studies have remained limited because of the lack of a robust cell culture system to amplify this virus. A few years ago, a model mimicking the entry process of HCV lifecycle has been developed by pseudotyping retroviral particles with native HCV envelope glycoproteins. This model allowed the characterization of functional E1E2 envelope glycoproteins. The data obtained can now be confirmed with the help of a newly developed cell-culture system that allows efficient amplification of HCV (HCVcc). Here, we present the recent data that have been accumulated on the assembly of the functional HCV glycoprotein heterodimer.